Changes in serum hyaluronic acid (hyaluronic acid is also known in the art as hyaluronate and hyaluronan) have been correlated with certain diseases. For example, an increase in serum hyaluronic acid has been found in patients having hepatic dysfunctions, such as cirrhosis. Additionally, there is a correlation between the increase in serum hyaluronic acid and cancer and rheumatoid arthritis. Methods for the detection and quantitation of hyaluronic acid in serum and other biological fluids are, therefore, desirable.
Cartilage proteoglycan core protein has a hyaluronic acid binding region which specifically and reversibly binds to hyaluronic acid. Keratan sulfate is a glycosaminoglycan covalently linked to the cartilage proteoglycan core protein in several areas and is concentrated in the keratan sulfate-rich region near the hyaluronic acid binding region.
Several radioassays have been disclosed for measuring the amount of hyaluronate in biological samples. For example, the Pharmacia HA Test 50 (Pharmacia Diagnostics AB, Uppsala, Sweden) is a radiometric assay for the determination of hyaluronic acid using .sup.125 I-labelled hyaluronic acid binding proteins from bovine cartilage. See also, A. Tengblad, "Quantitative Analysis of Hyaluronate in Nanogram Amounts" Biochem. J. 185, 101-105 (1980).
In Thonar et al, "Quantification of keratan Sulfate in Blood as a Marker of Cartilage Catabolism" Arthritis and Rheumatism 28(12), 1367-1376 (1985), there is described an enzyme-linked immunosorbent-inhibition assay (ELISA) using a monoclonal antibody specific for keratan sulfate to quantify keratan sulfate present as single chains in adult human serum.
In Delpech et al, "Immunoenzymeassay of the Hyaluronic-Hyaluronectin Interaction: Application to the Detection of Hyaluronic Acid in Serum of Normal Subjects and Cancer Patients" Anal Biochem 149, 555-565 (1985), the authors describe a method for investigating the binding of a hyaluronic acid-binding glycoprotein, hyaluronectin, extracted from the human brain, to hyaluronic acid using an enzyme-linked immunosorbent assay technique. This reference states the hyaluronectin is a protein component from the human brain which combines in vitro with hyaluronic acid but not with other glycosaminoglycans.
Prior to the present invention, there was no non-isotopic method for measuring the amount of hyaluronate in biological samples using cartilage proteoglycan.